Thermus thermophilus TMY isolated from silica scale taken from a geothermal power plant.

AIMS: To identify an extreme thermophile, strain TMY, isolated from silica scale from the geothermal electric power plant and to examine microdiversity of Thermus thermophilus strains. MATERIALS AND RESULTS: The isolated strain TMY was identified by morphological, biochemical and physiological tests. Phylogenetic comparison of the strain and other Thermus strains with 16S rDNA analysis, RAPD and ERIC-PCR fingerprinting were performed. Strain TMY was closely related to strain which was isolated from a hot spring in New Zealand and shown to belong to the Japanese Thermus cluster. However, there were considerable genetic differences between strain TMY and other Thermus species using DNA fingerprinting. CONCLUSIONS: Based on morphological, physiological and genetic properties, strain TMY could be a strain of T. thermophilus. The distinct properties of strain TMY suggest that microdiversity of T. thermophilus strains should be considered. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this study have demonstrated genetic diversity within T. thermophilus strains, which were previously masked by an almost identical 16S rDNA sequence. RAPD and ERIC-PCR could be potential methods for distinguishing between Thermus strains.

Association between the vanB2 glycopeptide resistance operon and Tn1549 in enterococci from France.

Linkage between the vanB2 gene cluster and transposon Tn1549 was found in clonally unrelated VanB-type vancomycin-resistant Enterococcus spp. strains isolated in France. The transposon was chromosomally located or plasmid borne.

[Serological cross-reaction among Bartonella henselae, Chlamydia pneumoniae and Coxiella burnetii by indirect fluorescence antibody method].

We studied the serological cross-reactions among Bartonella henselae, Chlamydia pneumoniae and Coxiella burnetii by indirect fluorescence antibody (IFA) method, using sera from 8 patients with cat scratch disease (CSD), 13 patients with C. pneumoniae infection and 12 patients with acute Q fever. B. henselae IgG antibody was negative in 13 patients with C. pneumoniae infection, and was positive in 3 (titers being 1:64) of 12 patients with Q fever, whereas B. henselae IgM antibody was negative in all the patients with C. pneumoniae infection or Q fever. C. burnetii IgG antibody was removed by absorption of these 3 sera with C. burnetii antigens, whereas B. henselae IgG antibody did not change. C. pneumoniae IgG antibody was positive in 3 (titers being 1:125 in two, 1:32 in one) of 8 patients with CSD. Both C. pneumoniae and B. henselae IgG antibody titers were significantly reduced by absorption of these 3 sera with B. henselae antigens. C. burnetii IgG or IgM antibodies were negative in all patients with CSD. In conclusion, no serological cross-reaction between B. henselae and C. burnetii was observed. On the other hand. B. henselae IgG antibody cross-reacted to C. pneumoniae antigens, whereas C. pneumoniae IgG antibody did not cross-react to B. henselae antigens. Our findings suggest that determination of B. henselae IgG or IgM antibodies were not influenced by C. pneumoniae and C. burnetii antigens.

Increased expression of transforming growth factor-beta1 in small airway epithelium from tobacco smokers and patients with chronic obstructive pulmonary disease (COPD).

Tobacco smoke is believed to cause small airway disease and then chronic obstructive pulmonary disease (COPD), but the molecular mechanisms by which small airway obstruction occurs remain unknown. To study the gene expression levels of transforming growth factor (TGF)-beta1, a potent fibrogenic factor, in small airway epithelium from smokers and patients with COPD, we harvested highly pure samples of epithelial cells from small airways under direct vision by using an ultrathin bronchofiberscope BF-2.7T (outer diameter 2.7 mm with a biopsy channel of 0.8 mm in diameter). The expression levels of TGF-beta1 were evaluated by reverse transcription-polymerase chain reaction (RT-PCR). The mRNA levels of TGF-beta1 corrected by beta-actin transcripts were significantly higher in the smoking group and patients with COPD than those in nonsmokers (p < 0.01). Furthermore, among smokers and patients with COPD, TGF-beta1 mRNA levels correlated positively with the extent of smoking history (pack-years) and the degree of small airway obstruction as assessed by measurements of flow-volume curves. Immunocytochemistry of the cells demonstrated more intense stainings for TGF-beta1 in samples from smokers and patients with COPD than from nonsmokers. Spontaneously released immunoreactive TGF-beta1 levels from cultured epithelial cells were more elevated in subjects with a history of smoking and patients with COPD than in nonsmokers. Our study showed a close link between smoking and expression of TGF-beta1 in small airways. Our results also suggested that small airway epithelial cells might be involved in obstructive changes found in smokers and patients with COPD.

Growth factors, extracellular matrix components and cell adhesion molecules Warthin's tumor.

We studied expressions of various growth factors, their receptors, cell adhesion molecules and extracellular matrix components in Warthin's tumor of the salivary gland with immunohistochemistry and reverse transcriptase-polymerase chain reaction (RT-PCR). Various growth factors and their receptors, such as transforming growth factor-alpha (TGF-alpha), heparin-binding epidermal growth factor-like growth factor (HB-EGF), TGF-beta2, TG-beta3, insulin-like growth factor (IGF)-I and -II, vascular endothelial growth factor (VEGF), EGF receptor (EGFR), erb-B4, TGF-betaRI and II, Flt and Flk-1 and IGF receptor Ibeta, were found in epithelial cells and/or in some lymphoid cells. Fibronectin, laminin, collagen type IV and tenascin were found in stroma of the lymphoid tissue. Integrins such as alpha3beta1 and beta3, Thy-1, CD44 and VCAM-1 were also expressed in epithelial and/or lymphoid cells. These various proteins may interact and regulate the proliferation and cell attachment of both epithelial and lymphoid components in this unique tumor.

Cluster analysis on multiple drugs susceptibility supplements genotyping of methicillin-resistant Staphylococcus aureus.

OBJECTIVE: To evaluate the typing power of cluster analysis of antimicrobial susceptibility. METHODS: Results of pulsed-field gel electrophoresis in 71 strains of methicillin-resistant Staphylococcus aureus were compared with cluster analysis of the diameter of growth inhibition in 11 drugs. Subjects were a consecutive series of patients (n = 71) from the wards and outpatient units of a community teaching hospital. RESULTS: The cluster analysis took 2 to 3 seconds once the data were entered into a computer. The sensitivity, specificity, and accuracy of the cluster analysis were 76.3%, 58.3%, and 73.2%, respectively, using genotyping as the reference. CONCLUSIONS: The cluster analysis offered real-time epidemiologic data at minimal cost and labor, warranting its cost-effective role.

Identification of the universal cofactor (auxilin 2) in clathrin coat dissociation.

Uncoating of clathrin-coated vesicles in neuronal cells requires hsc70 in concert with the cofactor auxilin which contains a J-domain as well as a domain with homology to dual specific phosphatases and tensin, known as PTEN. The question of whether an analogous factor operates in other cell types has until now remained unanswered. Here we show that it is the recently discovered and widely expressed cyclin G-associated protein kinase which fulfils the function of neuronal auxilin in hsc70-mediated clathrin coat dissociation. GAK possesses a J-domain, which stimulates the hsc70 ATPase, it competes with auxilin for clathrin binding and at sufficiently high concentrations acts as a clathrin assembly protein. Moreover, GAK binds to the gamma- and alpha-appendage domains of the adaptor proteins AP-1 and AP-2 in vitro and phosphorylates their medium chains. Cells that transiently overexpress GAK are impaired in respect of receptor-mediated endocytosis. In transfected cells clathrin is dislodged from coated pits/vesicles and co-localizes with GFP-GAK in the form of large aggregates. The cellular distribution of membrane-associated adaptors was unaffected by overexpression of GAK. Our results point to a hsc70/auxilin-based uncoating system as a ubiquitous feature of eukaryotic cells.

Structure-activity relationships for the Ca2+-releasing activity of 6-hydroxy-beta-carboline analogues in skeletal muscle sarcoplasmic reticulum-the effects of halogen substitution at C-5 and C-7.

This study of structure-activity relationships of 6-hydroxy-beta-carboline analogues has been performed on the basis of quantitative measurement of Ca2+-releasing activity in the sarcoplasmic reticulum of skinned fibres of skeletal muscle. Substitution of halogens for hydrogens at the C-5 and C-7 positions and further introduction of a methyl group into the N-9 position of 6-hydroxy-beta-carboline resulted in Ca2+-releasing activity. The 50% effective concentrations of 5,7-dibromoeudistomin D, 5,7-dichloroeudistomin D, 5,7-diiodoeudistomin D, 9-methyl-5,7-dibromoeudistomin D, 9-methyl-5,7-dichloroeudistomin D, 9-methyl-5,7-diiodoeudistomin D, and caffeine were 5.6 x 10(-6), 6.3 x 10(-6), 7.8 x 10(-6), 2.1 x 10(-6), 2.0 x 10(-5), 3.7 x 10(-5), and 4.7 x 10(-4) M, respectively, indicating that these analogues are 10-200 times more potent than caffeine. Substitution of bromine by chlorine or iodine at the C-5 and C-7 positions markedly reduced the activity of the analogues with a methyl group at the N-9 position. These results suggest that halogens at the C-5 and C-7 positions in the beta-carboline skeleton are essential for Ca2+-releasing activity and that an N-9 methyl group also affects the activity of these analogues. Thus, these 6-hydroxy-beta-carboline analogues might become powerful tools for studying the molecular mechanism of Ca2+ release in the sarcoplasmic reticulum.

Increased expression of inflammatory mediators in small-airway epithelium from tobacco smokers.

To study the inflammatory responses of small-airway epithelium in smokers, we harvested enough living epithelial cells (1.97 x 10(6) +/- 0.74 x 10(6)) with a new ultrathin fiberscope from the very peripheral airways of 22 current smokers and 17 subjects who never smoked after informed consent was obtained. The cells were keratin positive and composed mainly of nonciliated cells. The expression levels of inflammatory markers [interleukin (IL)-8 and intercellular adhesion molecule (ICAM)-1] were evaluated with RT-PCR. The magnitude of the mRNA levels corrected by beta-actin transcripts of IL-8 and ICAM-1 was significantly higher in the smokers than in the nonsmokers (P < 0.001). Furthermore, among current smokers, IL-8 mRNA levels correlated positively with the extent of smoking history [in pack. years (packs/day x no. of years of smoking); r = 0.754, P < 0.001]. Spontaneously released IL-8 and soluble ICAM-1 levels (n = 12) from cultured epithelial cells were elevated in subjects with a smoking history than in those without it (IL-8, 1,580 +/- 29.6 vs. 354 +/- 39.4 pg. 10(6) cells(-1). 24 h(-1); P < 0.001; soluble ICAM-1, 356.0 +/- 45.9 vs. 112.9 +/- 12.9 pg. 10(6) cells(-1). 24 h(-1); P < 0.01 by Student's t-test ). In contrast, the epithelial cells from the main bronchi did not show such differences between smokers and nonsmokers. Our study highlighted a close link between smoking and the expression of inflammatory mediators such as IL-8 and ICAM-1 in small airways. Our results also suggested that this new ultrathin bronchofiberscope promised a good approach for the evaluation of cellular changes in the small airways.

Association of Vibrio cholerae O1 with the cyanobacterium, Anabaena sp., elucidated by polymerase chain reaction and transmission electron microscopy.

It has been hypothesized that Vibrio cholerae is an autochthonous flora of the estuarine and brackish water environment. Zooplankton and phytoplankton have been considered as possible reservoirs. The present study was carried out in microcosms to confirm the role of a cyanobacterium, Anabaena sp., as a reservoir of V. cholerae O1 using culture, polymerase chain reaction (PCR) and immunoelectron microscopy. Survival of culturable V. cholerae in microcosms was monitored by using tellurite taurocholate gelatin agar. Culturable V. cholerae were detected for up to 1 h in association with Anabaena sp. from a microcosm. However, viable but nonculturable (VBNC) V. cholerae O1 were detected for up to 25 months using PCR and immunoelectron microscopy. Results also showed that VBNC V. cholerae can multiply and maintain their progeny in the mucilaginous sheath of Anabaena sp. This is the first time that PCR and immunoelectron microscopy have been used to detect nonculturable V. cholerae in association with Anabaena sp. This study further clarifies the role of Anabaena sp. as a possible reservoir of cholera.

Subtyping of Haemophilus influenzae strains by pulsed-field gel electrophoresis.

A total of 200 isolates of Haemophilus influenzae were analyzed by serotyping, biotyping, and pulsed-field gel electrophoresis (PFGE). A total of 178 epidemiologically unrelated strains of H. influenzae demonstrated a variety of genome patterns by PFGE, and 165 genotypes were thus obtained in this study. PFGE typing proved to have a much stronger discriminatory power than either serotyping or biotyping. Six serotype b strains were all classified into discrete genotypes. A PFGE analysis of 18 strains obtained from the nasopharynx, blood, and cerebrospinal fluid of patients with meningitis also supported the hypothesis that invasive H. influenzae disseminates from the nasopharynx to the bloodstream and then subsequently to other body sites. PFGE typing of 10 other strains isolated from household contacts of patients with H. influenzae infection revealed that the strain that caused the H. influenzae infection often colonized the nasopharynges of household contacts. Our findings suggest that PFGE analysis is useful for the epidemiological study of H. influenzae infection, even when the invasive disease is caused by serotype b strains.

Morphological behavior of acidic and neutral liposomes induced by basic amphiphilic alpha-helical peptides with systematically varied hydrophobic-hydrophilic balance.

Lipid-peptide interaction has been investigated using cationic amphiphilic alpha-helical peptides and systematically varying their hydrophobic-hydrophilic balance (HHB). The influence of the peptides on neutral and acidic liposomes was examined by 1) Trp fluorescence quenched by brominated phospholipid, 2) membrane-clearing ability, 3) size determination of liposomes by dynamic light scattering, 4) morphological observation by electron microscopy, and 5) ability to form planar lipid bilayers from channels. The peptides examined consist of hydrophobic Leu and hydrophilic Lys residues with ratios 13:5, 11:7, 9:9, 7:11, and 5:13 (abbreviated as Hels 13-5, 11-7, 9-9, 7-11, and 5-13, respectively; Kiyota, T., S. Lee, and G. Sugihara. 1996. Biochemistry. 35:13196-13204). The most hydrophobic peptide (Hel 13-5) induced a twisted ribbon-like fibril structure for egg PC liposomes. In a 3/1 (egg PC/egg PG) lipid mixture, Hel 13-5 addition caused fusion of the liposomes. Hel 13-5 formed ion channels in neutral lipid bilayer (egg PE/egg PC = 7/3) at low peptide concentrations, but not in an acidic bilayer (egg PE/brain PS = 7/3). The peptides with hydrophobicity less than Hel 13-5 (Hels 11-7 and Hel 9-9) were able to partially immerse their hydrophobic part of the amphiphilic helix in lipid bilayers and fragment liposome to small bicelles or micelles, and then the bicelles aggregated to form a larger assembly. Peptides Hel 11-7 and Hel 9-9 each formed strong ion channels. Peptides (Hel 7-11 and Hel 5-13) with a more hydrophilic HHB interacted with an acidic lipid bilayer by charge interaction, in which the former immerses the hydrophobic part in lipid bilayer, and the latter did not immerse, and formed large assemblies by aggregation of original liposomes. The present study clearly showed that hydrophobic-hydrophilic balance of a peptide is a crucial factor in understanding lipid-peptide interactions.

BASH, a novel signaling molecule preferentially expressed in B cells of the bursa of Fabricius.

The bursa of Fabricius is a gut-associated lymphoid organ that is essential for the generation of a diversified B cell repertoire in the chicken. We describe here a novel gene preferentially expressed in bursal B cells. The gene encodes an 85-kDa protein, designated BASH (B cell adaptor containing SH2 domain), that contains N-terminal acidic domains with SH2 domain-binding phosphotyrosine-based motifs, a proline-rich domain, and a C-terminal SH2 domain. BASH shows a substantial sequence similarity to SLP-76, an adaptor protein functioning in TCR-signal transduction. BASH becomes tyrosine-phosphorylated with the B cell Ag receptor (BCR) cross-link or by coexpression with Syk and Lyn and associates with signaling molecules including Syk and a putative chicken Shc homologue. Overexpression of BASH results in suppression of the NF-AT activation induced by BCR-cross-linking. These findings suggest that BASH is involved in BCR-mediated signal transduction and could play a critical role in B cell development in the bursa.

9-Methyl-7-bromoeudistomin D induces Ca2+ release from cardiac sarcoplasmic reticulum.

9-Methyl-7-bromoeudistomin D (MBED), the most powerful caffeine-like releaser of Ca2+ from skeletal muscle sarcoplasmic reticulum, induced Ca2+ release from the cardiac sarcoplasmic reticulum. MBED (5 microM) and caffeine (1 mM) caused rapid Ca2+ release from the fragmented cardiac sarcoplasmic reticulum in a Ca2+ electrode experiment. [3H]MBED bound to a single class of high-affinity binding sites in cardiac sarcoplasmic reticulum membranes (Kd = 150 nM). These results suggest that MBED binds to a specific binding site on cardiac sarcoplasmic reticulum membranes to induce Ca2+ release from the cardiac sarcoplasmic reticulum. Thus, MBED is a useful probe for characterizing Ca2+ release the channels not only in skeletal sarcoplasmic reticulum but also in cardiac sarcoplasmic reticulum.

[Structure of the bacterial cell wall].

The fine structure of the cell walls of Gram-positive and -negative bacteria were determined by electron microscopy with the new technique of freeze substitution method, and analysed the cell wall structure of Staphylococcus aureus in detail. The surface of Staphylococcal cell wall was covered with a fuzzy coat consisting of fine fibers or electron-dence mass. This coat was completely removed after extraction of teichoic acid from the cell wall with trichloroacetic acid treatment, but was not affected by sodium dodecyl sulfate or trypsin treatment. It was suggested that many amount of teichoic acid was located on the surface of the cell wall and less inside the cell wall. The capsule of strain Smith diffuse was assumed to play the role as the barrier protected from the penetration of antibody against teichoic acid.

Surface characteristics of gram-negative and gram-positive bacteria in an atomic force microscope image.

Bacterial images can be obtained rather easily with an atomic-force microscope (AFM) in the magnification range of 5,000 to 30,000 times without any pretreatment of the specimens for such observations as chemical fixation, dehydration or staining. The bacterial shapes or the presence of flagella can be clearly recognized in these magnification ranges. In addition, we were also able to distinguish between gram-negative and gram-positive bacteria based on the specific wavy surface appearance of the former. AFM could thus be a useful tool for the identification of bacteria in the resolution range between electron and light microscopy.

Spirochaete-like swimming mode of Campylobacter jejuni in a viscous environment.

The swimming patterns of Campylobacter jejuni in environments of low and high viscosity were examined by a video tracking method. In media of low viscosity, C. jejuni swam with an average velocity of 39.3 microm/s with frequent changes in direction. The velocity of C. jejuni increased in a medium at a little higher viscosity than that of a low viscosity buffer. In addition to this, C. jejuni showed a second increase of velocity in media of a high viscosity of about 40 centipoise. The swimming patterns at these two velocity peaks were compared. In the second peak the wild-type C. jejuni exhibited repeated back and forth swimming patterns which were more like the swimming pattern of spirochaetes than that of monotrichous bacteria. Thus C. jejuni may presumably use a different swimming mode in media of high viscosity than the original swimming mode mediated by the propelling force of the flagella. The spiral shape of this bacterium like that of spirochaetes may strongly influence its swimming ability in media of high viscosity such as the mucous layer of the intestinal tract.

Displacement of gold marker in immunoelectron microscopy of human respiratory cilia.

Preembedding immunogold electron microscopy was performed to evaluate the position of outer arm dynein heavy chains in normal human respiratory cilia. Anti-dynein antibody (AD2), which is specific for sea urchin sperm flagellar dynein heavy chains, was used as primary antibody. Direct cross-sections of cilia were selected, and the distance between the center of a cilium and the center of a colloidal gold particle attached to the cilium (X) was measured. The distance between the center of a cilium and the farthest edge of an outer dynein arm of the cilium was measured by ordinary electron microscopy (Yo) and by immunoelectron microscopy (Yi). X was significantly longer than Yo and Yi. If it is assumed that the structure of respiratory cilia is dense and that antibodies are located at the outer side of the actual position of the heavy chains, then the average distance difference of approximately 90-120 A may represent the length of two conjugated antibodies. This length should be kept in mind when performing immunoelectron microscopy. The data suggest that AD2 recognizes the outer arm dynein heavy chains of normal human respiratory cilia.

Synthesis of oligodeoxyribonucleotide containing novel C-5 reactive 2'-deoxyuridine derivative and its functional modification via post-synthetic technique.

2'-Deoxyuridine derivatives bearing an activated ester at C-5 position were synthesized and was examined their use for the preparation of modified oligodeoxyribonucleotides (ODNs) by a post-modification method. The ODNs containing cyanomethyl ester at C-5 position of the deoxyuridine residue reacted easily with a primary amine of several functional molecules under the mild condition to give the corresponding modified ODNs.

A structural analysis of the regularly arranged porin on the outer membrane of Campylobacter jejuni based on correlation averaging.

A negatively stained electron micrograph of regularly arranged porin proteins of Campylobacter jejuni on the isolated outer membrane of bacteria was analyzed in detail by the correlation averaging method using a computer-assisted program. The results showed that the porin of C. jejuni had a trimeric structure separated by about 10.4 +/- 0.15 nm. In addition, the pores in the trimers were also separated by about 4.3 +/- 0.1 nm.